This is an R21 proposal to investigate the biochemistry and genetics of Phospholipase D4, a novel gene of unknown function with homology to classical phospholipase D genes. Related family member proteins, such as PLD1 and PLD2 convert phosphatidyl choline (PC) to phosphatidic acid, releasing choline, a reaction that has effects on both cell signaling and membrane dynamics. PLD4 carries key active site amino acids of PLD1 and 2, but lacks classical phospholipase D activity. More distantly related family members have nuclease or DNA-tyrosine phosphodiesterase activity. Particularly close homologues to PLD4 are found in pox viruses. PLD4 is expressed in a variety of antigen presenting cells, including B cells, with highest levels in endosomal compartments of myeloid cells, especially plasmacytoid dendritic cells. Very low, but detectable, levels of expression are also seen in T cells. We have recently generated a conditional knockout allele of Pld4 and have begun the analysis of Pld4 germline null mice. At three months of age these mice already exhibit evidence of multiorgan inflammation, splenomegaly, possible defects in B cell development and thymic selection, and excess numbers of activated and memory B and T cells. We hypothesize that PLD4 plays a role in antigen presentation through the regulation of endosomal function. To understand how Pld4 deficiency causes the immunological phenotype we propose to characterize mice lacking Pld4 in selected tissues and cell types. A second goal is to determine the enzymatic activity of PLD4 using a variety of biochemical approaches, including "lipidomics" screens. PUBLIC HEALTH RELEVANCE: This is a R21 exploratory grant proposal to elucidate the function of a novel gene called Pld4. We have mutated this gene in mice and found that the mutants develop severe inflammation in many organs, including lung, salivary gland, liver and kidney, suggesting dysregulation of the immune system. However, the key cell types in which Pld4 must function to avoid disease are not known, nor is it clear what biochemical role the Pld4 gene product plays, though it is implicated in the regulation or formation of small vesicles within cells. This project will attempt to shed light on these issues. Because human beings carry a nearly identical gene to that of mouse, our studies have direct relevance to the understanding of the human gene and possible diseases caused by Pld4 deficiency or dysregulation.